Thrombin is a multifunctional protease that regulates several key biological processes. For example, thrombin is among the most potent of the known platelet activators. In addition, thrombin is essential for the cleavage of fibrinogen to fibrin to initiate clot formation. These two elements are involved in normal hemostasis but in atherosclerotic arteries can initiate the formation of a thrombus, a major factor in pathogenesis of vasoocclusive conditions such as myocardial infarction, unstable angina, nonhemorrhagic stroke and reocclusion of coronary arteries after angioplasty or thrombolytic therapy. Thrombin is also a potent inducer of smooth cell proliferation and may therefore be involved in a variety of proliferative responses such as restenosis after angioplasty and graft-induced atherosclerosis. In addition, thrombin is chemotactic for leukocytes and may therefore play a role in inflammation. (Hoover, R. J., et al. Cell (1978) 14:423; Etingin, O. R., et al., Cell (1990) 61:657.) These observations indicate that inhibition of thrombin formation or inhibition of thrombin itself may be effective in preventing or treating thrombosis, limiting restenosis and controlling inflammation.
The formation of thrombin is the result of the proteolytic cleavage of its precursor prothrombin at the Arg-Thr linkage at positions 271-272 and the Arg-Ile linkage at positions 320-321. This activation is catalyzed by the prothrombinase complex, which is assembled on the membrane surfaces of platelets, monocytes, and endothelial cells. The complex consists of Factor Xa, which is secreted by the liver as a 58 kd precursor and is converted to the active form, Factor Xa, in both the extrinsic and intrinsic blood coagulation pathways. It is known that the circulating levels of Factor X, and of the precursor of Factor Va, Factor V, are on the order of 10.sup.-7 M. There has been no determination of the levels of the corresponding active Factors Va and Xa.
The complete amino acid sequences of human Factor X and Factor Xa are known, and are as described by Davie, E. W., in Hemostasis and Thrombosis, Second Edition, R. W. Coleman et al., eds. (1987) p. 250. Factor X is a member of the calcium ion binding, gamma carboxyglutamyl (Gla)-containing, vitamin K dependent, blood coagulation glycoprotein family, which also includes Factors VII and IX, prothrombin, protein C and protein S (Furie, B., et al., Cell (1988) 53:505).
The mature Factor X protein is preceded by a 40-residue pre-pro leader sequence which is removed during intracellular processing and secretion. The mature Factor X precursor of Factor Xa is then cleaved to the two-chain form by deletion of the three amino acids RKR between the light chain C-terminus and activation peptide/heavy chain N-terminus. Finally, the two chain Factor X is converted to Factor Xa by deletion of the 52 amino acid "activation peptide" sequence generating a light chain of 139 residues and a heavy chain of 254 residues. These are linked through a single disulfide bond between position 128 of the light chain and position 108 of the heavy chain. The light chain contains the Gla domain and an epidermal growth factor-like domain; the protease activity resides in the heavy chain and involves the histidine at position 42, the aspartic at position 88, and a serine at position 185.
Bovine Factor X has also been studied, and the amino acid sequence of bovine Factor X has been reported by Titani, K., Proc Natl Acad Sci USA (1975) 72:3082-3086. The activation peptide of bovine Factor X is 51 amino acids long and contains almost equal masses of polypeptide and carbohydrate (Discipio, R. G., et al., Biochemistry (1977) 16:5253).
There are two known pathways for the activation of the two-chain Factor X in vivo. Activation must occur before the protease is incorporated into the prothrombinase complex (Steinberg, M., et al., in Hemostasis and Thrombosis, Coleman, R. W., et al., eds. (1987) J. B. Lippencott, Philadelphia, Pa., p. 112). In the intrinsic pathway, Factor X is cleaved to release the 52-amino acid activation peptide by the "tenase" complex which consists of Factor IXa, Factor VIIIa and calcium ions assembled on cell surfaces. In the extrinsic pathway, the cleavage is catalyzed by Factor VIIa which is bound to a tissue Factor on membranes. Factor X may also be converted to Factor Xa by in vitro cleavage using a protease such as that contained in Russel's viper venom. This protease is described by DiScipio, R. G., et al., Biochemistry (1977) 6:5253.
Factor X variants exist that are differentially activated in the extrinsic versus intrinsic pathway (Fair, D. S. et al., J Clin Invest (1979) 648:884-894). For example, Factor X "Vorarlberg" has 15% of the activity of normal Factor X with respect to the extrinsic pathway but 75% of normal Factor X with respect to the intrinsic pathway (Watzke, H. H. et al., J Biol Chem (1990) 265:11982-11989).
Clearly, the action of thrombin in platelet aggregation and clot formation could be prevented by suitable manipulation of factor X/Xa, in view of the essential role of factor Xa in the formation of thrombin from its precursor.
The activity of Factor Xa in effecting the conversion of protrombin to thrombin is dependent on its inclusion in the prothrombinase complex. Therefore, one approach to this manipulation has been directed to preventing the participation of factor Xa in the thrombinase complex. The formation of the prothrombinase complex (which is 278,000 fold faster in effecting the conversion of prothrombin to thrombin than Factor Xa in soluble form) has been studied (Nesheim, H. E., et al., J Biol Chem (1979) 254:10952). These studies have utilized the active site-specific inhibitor, dansyl glutamyl glycyl arginyl (DEGR) chloromethyl ketone, which covalently attaches a fluorescent reporter group into Factor Xa. Factor Xa treated with this inhibitor lacks protease activity, but is incorporated into the prothrombinase complex with an identical stoichiometry to that of Factor Xa and has a dissociation constant of 2.7.times.10.sup.-6 M (Nesheim, M. E., J Biol Chem (1981) 256:6537-6540; Skogen, W. F., et al., J Biol Chem (1984) 256:2306-2310; Krishnaswamy, S., et al., J Biol Chem (1988) 263:3823-3824; Husten, E. J., et al., J Biol Chem (1987) 262:12953-12961).
Other approaches to inhibition of Factor Xa include the use of lipoprotein-associated coagulation inhibitor (LACI), now called tissue factor pathway inhibitor (TFPI) (Girard, T. J., et al., Nature (1989) 338:518; Girard, T. J., et al., Science (1990) 248:1421), leech-derived antistatin (Dunwiddie, C. T., et al., J Biol Chem (1989) 264:16694), and tick-derived TAP (Waxman, L., et al., Science (1990) 248:593). Alternatively, agents which inhibit the vitamin K-dependent Gla conversion enzyme, such as coumarin, have been used. None of these approaches have proved satisfactory due to lack of specificity, the large dosage required, toxic side effects, and the long delay in effectiveness.
PCT publication US 91/06337 discloses an additional approach wherein the active site of Factor Xa is modified to prevent its enzymic activity while retaining the ability to form the prothrombinase complex.
The invention approach is directed to the inhibition of the conversion of Factor X to its active form, Factor Xa. Specifically, this mode of inhibition is directed to manipulation of the glycosylation residues associated with Factor X.
Bovine, but not human, Factor X has been subjected to detailed studies with respect to its glycosylation patterns. The heavy chain of bovine Factor X contains N-linked glycosylation at residue 36 and O-linked glycosylation at residue 300 (Mizouchi, T. et al., J Biol Chem (1980) 255:3526-3531). While the glycosylation pattern of human Factor X is not known, it has been noted that the activation peptide contains two potential N-linked glycosylation sites at positions 39 and 49 (Davie, E. W., in "Hemeostasis and Thrombosis", Second Edition, R. W. Coleman eds. (1987) p. 250). In addition, serine-linked sugar residues have been reported on the first epidermal growth factor-like domain of the related bovine factor IX (Hase, S. et al., J Biol Chem (1990) 265:1858-1861); analogously, the EGF-like domains on human Factor X light chain may contain O-linked sugars.
Accordingly, the invention offers an alternative approach to inhibit the conversion of Factor X to Factor Xa, thus preventing formation of an active prothrombinase complex.